This invention relates to: a new DNA sequence ("the bt4 gene") from the genome of the strain Bacillus thuringiensis var. aizawai. HD-68 (the "Bt HD-68 strain") which is publicly available from the Agricultural Research Culture Collection, Northern Region Research Center, 1815 North University Street, Peoria, Ill. 61604, U.S.A. The bt4 gene encodes a 132 kDa protein (the "Bt4 protoxin"). This invention additionally relates to a new DNA sequence (the "bt18 gene") from the genome of the strain Bacillus thuringiensis var. darmstadiensis HD-146 (the "Bt HD-146 strain") which is also publicly available from the Agricultural Research Culture Collection, U.S.A. The Bt18 gene encodes a 130 kDa protein (the "Bt18 protoxin").
This invention also relates to a 60 kDa protein (the "Bt4 toxin") and a 62 kDa protein (the "Bt18 toxin") which can be obtained by trypsin digestion of the Bt4 protoxin and Bt18 protoxin, respectively. The Bt4 toxin is the active ingredient in the crystallized protoxin produced by the Bt HD-68 strain, with a high activity against Lepidoptera species, such as Manduca sexta and Spodoptera species. The Bt18 toxin is the active ingredient in the crystallized protoxin produced by the Bt HD-146 strain, with a high activity against Lepidoptera species from the Noctuidae family such as Spodoptera species, as well as other Lepidoptera such as Manduca sexta.
As is the case for other B. thuringiensis ("Bt") crystal proteins (Hofte et al, 1988), when the crystalline Bt4 or Bt18 protoxin is ingested by insect larvae, it is solubilized and processed in the insect's midgut, releasing the Bt4 toxin or Bt18 toxin, respectively. In this regard, Hofte et al (1988) has generally described three types of toxin-producing Lepidoptera-specific B. thuringiensis having the following characteristics:
Type A (consisting of 3 subtypes) producing a protoxin of 130 kDa and a toxin of 60 kDa which is toxic against Manduca sexta and Pieris brassicae. PA1 Type B producing- a protoxin of 133 kDa and a 55 kDa toxin which is toxic against Pieris brassicae. PA1 Type C producing a 135 kDa protoxin and a 63 kDa trypsin-activated toxin, showing insecticidal activity against Spodoptera littoralis and Mamestra brassicae. PA1 1) all or an insecticidally effective part of the bt4 or bt18 gene encoding all or an insecticidally effective part of the respective Bt4 or Bt18 protoxin, preferably a truncated part of the bt4 or bt18 gene ("the truncated bt4 or bt18 gene") encoding just the respective Bt4 or Bt18 toxin; PA1 2) a promoter suitable to direct transcription of all or part of the bt4 or bt18 gene in the plant cell; and PA1 3) suitable 3' transcription regulation signals for expressing all or part of the bt4 or bt18 gene in the plant cell.
This invention further relates to a chimaeric gene that can be used to transform a plant cell and that contains the following, operably linked, DNA sequences:
This chimaeric gene is hereinafter referred to as the "bt4 or bt18 chimaeric gene". Preferably, the plant cell is transformed with a bt4 or bt18 chimaeric gene comprising the truncated bt4 or bt18 gene, together with a selectable marker gene, such as the neo gene encoding neomycin phosphotransferase II or NPTII (Reiss et al, 1984 ), fused with the truncated bt4 or bt18 gene as a bt4-neo or bt18-neo hybrid gene encoding a Bt4 toxin - NPTII or Bt18 toxin-NPTII fusion protein.
This invention still further relates to a plant that is regenerated from the transformed cell and that is resistant to Lepidoptera, particularly Sphingidae such as Manduca sexta and Noctuidae such as Spodoptera species which are major pests of economically important crops such as cotton, corn, soybean, alfalfa, tomato, tobacco, sugarbeet and other vegetables.
This invention yet further relates to: a method of locating the C-terminal end of the minimum toxic part or core of a B. thuringiensis protoxin which is like the Bt4 or Bt18 protoxin and is hereinafter referred to as a "Bt4-like protoxin"; a plant cell and plant transformed with a DNA sequence encoding a toxic part of a Bt4-like protoxin having a minimum-length C-terminal end; and a probe for identifying such a DNA sequence.